Research Summary The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980?s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, responding to PAR-16-176, we propose to conduct studies to validate a lesser known model of osteosarcoma in rats by a comprehensive histologic, biologic and proteomic comparison to human osteosarcoma, with exploration of a novel molecular cell death imaging method assessing standard and/or targeted therapeutics. We hypothesize that this model is pre-clinically relevant for the study of pediatric osteosarcoma for the following reasons. This is an immunocompetent, outbred Sprague-Dawley, syngeneic rat model with implanted UMR106 osteosarcoma cell line, with orthotopic tibial implants in young male and female rats, who develop reproducible primary and metastatic pulmonary tumors, using limb amputation to control the incidence of pulmonary metastasis. Histologically, primary and metastatic osteosarcomas in rats are very similar to human osteosarcoma. Rat OS expresses ErbB family kinases, which were inhibited by anti-ErbB2, offering this model as an opportunity to study this pathway in osteosarcoma. Using TMT labeling methods, we will perform a proteomic comparison between rat UMR106 osteosarcoma cell line and primary rat osteoblasts, with a second comparison between primary rat osteosarcoma and matched pulmonary metastasis. Rat proteomics data will be analyzed against published datasets on human osteosarcoma to identify common pathways and therapeutic targets and confirm the validity of the rat model of osteosarcoma. Next, we will explore SPECT/CT imaging with Tc99m-HYNIC-Annexin-V to quantify cell death in osteosarcoma using standard and novel therapeutic agents with longitudinal pulmonary metastasis assessments. We will assess a dose response with doxorubicin, followed by a separate study using rat anti-ErbB2 alone. ErbB2 is increased in UMR106 rat OS cells and anti-ErbB2 inhibits AKT phosphorylation. Using (rat anti-ErbB2) (7.16.4) in combination with doxorubicin, cell death in both tumors and hearts will be assessed simultaneously by in vivo SPECT cell death imaging, with follow up histologic and TUNEL comparison of the primary OS in the amputated leg. Importantly, rats will be longitudinally monitored up to 9 months for pulmonary metastases and off-target cardiotoxicity.